This invention relates to a monoclonal antibody against P-glycoprotein and its use in detecting drug resistant carcinoma.
Multidrug resistance in human cancer is a unique phenomenon and is commonly associated with an overexpression of the human multidrug resistant gene mdrl, which encodes an energy-dependent Mr 170,000 transmembrane protein, also known as P-glycoprotein. One biological function of P-glycoprotein is to transport some chemotherapeutic agents out of cancer cells, thereby conferring a drug resistant phenotype to cancer cells expressing P-glycoprotein. The recognition of the importance of a complexed drug resistant phenotype of broad spectrum in human cancer has prompted an extensive investigation of this phenomenon in vitro using drug resistant cell lines (Juranka, P. F. et al., P-Glycoprotein: multidrug-resistance and a super family of membrane-associated transport protein. FASEB J. 3: 2583-2592, 1989; Pastan, I. et al., Molecular manipulation of the multidrug transporter: a new role for transgenic mice. FASEB J. 5: 2523-2528, 1991; Gros, P. et al., Multidrug resistance: A novel class of membrane-associated transport protein is identified. Cancer Invest. 9: 563-569, 1991; Roninson, I. B. et al., Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells. Proc. Natl. Acad. Sci. USA 83: 4538-4542, 1986; Ling, V. et al., U.S. Pat. No. 4,837,306 and Canadian Patent 1,263,324.) All patents and other documents cited herein are incorporated herein by reference.
Immunological and molecular biology techniques have permitted the identification, isolation, and characterization of the mdrl gene, and its encoded P-glycoprotein. Recent study has revealed that P-glycoprotein represents a member of a large family of homologous membrane associated transport proteins, which are implicated in multidrug resistance and other diseases. Biochemically, P-glycoprotein is found in the plasma membrane enriched fraction, is glycosylated, exhibits ATPase activity, binds photoactivatable ATP, and drug analogs. The prototype P-glycoprotein is consisted of twelve transmembrane domains capable of forming six transmembrane loops, a cluster of putative N-linked glycosylation sites located between the first and second proposed transmembrane domain, and two predicted cytoplasmic ATP binding sites. Additionally, peptide portion of P-glycoprotein is composed of two highly symmetrical halves.
From an immunochemical point of view, this seemingly complex and large glycoprotein structure would represent at least a dozen of distinct antigenic determinants. Yet, only a handful of McAb directed against human P-glycoprotein have been generated (Kartner, N. et al., Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature (Lond.) 316: 820-823, 1985; Lathan, B. et al., Immunological detection of Chinese hamster ovary cells expressing a multidrug resistance phenotype. Cancer Res. 45: 45064-5069, 1985; Hamada, H. et al., Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cell as revealed by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 83: 7785-7789, 1986; Scheper, R. J. et al., Monoclonal antibody JSB-1 detects a highly conserved epitope on the P-glycoprotein associated with multi-drug resistance. Int. J. Cancer 42: 389-394, 1988; Meyers, M. B., et al., Characterization of monoclonal antibodies recognizing a Mr 180,000 P-glycoprotein: Differential expression of the Mr 180,000 and Mr 170,000 P-glycoproteins in multi-drug resistant human tumor cells. Cancer Res. 49: 3209-3214, 1989), in spite of the importance of multidrug resistance in pharmacology and therapy of cancer (Chabner, B. A. et al., Multidrug resistance: P-glycoprotein and its allies-the elusive foes. J. Natl. Cancer Inst. 81: 910-913, 1989; Dalton et al., Does P-glycoprotein predict response to chemotherapy, and if so, is there a reliable way to detect it? J. Natl. Cancer. Inst. 83: 80-84, 1991; Beck, W. T., Do anti-P-glycoprotein antibodies have a future in the circumvention of multidrug resistance? J. Natl. Cancer Inst. 83: 1364-1366, 1991). There have further been problems associated with the use of P-glycoprotein in detecting drug resistant cancer cells since biopsy and cell lysing and/or other complicated preparation has been required often taking up to one weeks time to complete an analysis and as long as 2-5 days, even when the procedures are rushed, e.g. by twenty-four hour staffing of the laboratory doing the test.